A molecular probe for high-resolution dual imaging of temperature and oxygen in vivo Sergei A. Vinogradov Project Summary The ability to image temperature gradients in highly spatially resolved manner would be invaluable for many areas of biomedical research. Optical measurements offer several important advantages, including minimal invasiveness, high spatial resolution in 3D (potentially limited by diffraction) and rapid response time. In addition to addressing basic questions of physiology, optical imaging of temperature would be highly useful in thermotherapy of cancer, diagnostics of local inflammation as well as in optimization of hypothermic treatment of brain injury and stroke. Furthermore, for quantitative imaging of oxygen distributions, independent non-invasive highly localized measurements of temperature are required, since calibrations of all oxygen measurement methods are intrinsically temperature-dependent. At the present time no probe exists that allows reliable optical imaging of temperature in tissue in vivo. Here we propose to develop the first molecular probe for imaging temperature concurrently with oxygen in vivo in 3D. The probe will be based on a novel chromophore capable of microsecond-scale emission occurring simultaneously from two well spectrally resolved excited electronic states, existing in thermal equilibrium with one-another. While the decay time will be sensitive to both temperature and oxygen, the ratio of the decay intensities will undergo changes only with temperature. The probe will be efficiently excitable by two-photon process, therefore allowing high-resolution imaging in 3D. It will be tested and validated by performing temperature (and oxygen) gradient measurements in the upper 0.5-1 mm layer of exposed rodent brain by means of two-photon phosphorescence lifetime microscopy (2PLM). We anticipate that the new probe will find applications in many areas, as all enzymatic reactions and as well as all detection methods depend on temperature.